S179: HNRNPK OVEREXPRESSION DRIVES NUCLEOLAR ABERRANCIES CAUSING RIBOSOMPATHIES
Authors: Pedro Aguilar Garrido, Maria Velasco Estevez, Miguel Ángel Navarro Aguadero, María Hernández Sánchez, Prerna Malaney, Marisa Hornbaker, Xiaorui Zhang, Marta Ibañez Navarro, Alejandra Ortiz-Ruiz, Álvaro Otero-Sobrino, Diego Megías, Manuel Pérez, Jesús Gómez, Gadea Mata, Orlando Dominguez, Osvaldo Grana, Eduardo Caleiras, Marta Isasa, Paloma Jimena de Andrés, Sandra Rodriguez, Raúl Torres, Oleksandra Sirozh, Vanesa Lafarga, Joaquín Martínez-Lopez, Sean Post, Miguel Gallardo
Journal: HemaSphere (2023)

Background: Protein biogenesis is a complex multistep process involving nucleoli and ribosomes. Aberrations in any step could lead to alterations in protein output. hnRNP K is an RNA binding protein (RBP) that process nascent RNAs into mature mRNAs and we found that its overexpression results in nuclear and nucleolar abnormalities, a decrease in transcription and potentially promotes an imbalance in ribosome biogenesis, leading to a decrease in protein translation. Aims: We aim to elucidate how hnRNP K dysregulation impact on HSC biology. Methods: To study the impact of hnRNP K overexpression in vivo, we developed an inducible tamoxifen mouse model activated 30-60 days after birth (HnrnpkTg-Ubc-creERT2). Survival was evaluated by Kaplan-Meier, and phenotype was described by symptoms, signs, CBC, bone marrow (BM) H/E, IHC and FCM analysis, and serum IL-6 ELISA. Hnrnpk overexpression was established in MEFs using CRISPR/SAM. MEFs RNA-Seq analysis was performed in a single read 85-base format and analyzed with DESeq2 and TMT-based deep proteome profiling was also performed. Both were GSEA preranked. Transcription and translation were tested using a Click-it RNA and an HPG Imaging kit respectively, and translation efficiency by polysome assay. Nucleolar aberrancies were analyzed by confocal microscopy evaluating Ncl and Fbl. Cell cycle FACS analysis (DAPI), senescence assays (β-galactosidase staining) and karyotyping were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB. Results:HnrnpkTg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Fig. A) and a reduction in lifespan (Fig. B). By CBC analysis, we observed the development of leukopenia, lymphopenia, anaemia and thrombocytopenia (Fig. C). Bone marrow H/E, IHC and FCM analysis showed a reduction of B220+ lymphoid cells and an increment in myeloid cells, with a reduction in CD34+ and Sca1+ HSCs (Fig. D). Moreover, we found higher senescent β-galactosidase expression in BM and more pro-senescent cytokine IL-6 in Hnrnpk overexpression mouse serum (Fig. E). To understand hnRNP K implication in BM failure phenotype in vivo, we generated Hnrnpk overexpressing cells (Fig. F). RNA-Seq from overexpressing Hnrnpk showed an upregulation in molecules related to G2/M checkpoint pathway (Fig. G) that was confirmed by FACS analysis showing and increment of arrested G2/M phase cells (Fig. H). Moreover, we showed a rise in senescence (β-galactosidase analysis), polyploidy and genomic instability (Fig I), linked to an increment in p21 and p16 levels (Fig. J). Then, we performed TMT proteomics analysis and noticed that Hnrnpk expression directly correlates with ribosome biogenesis alteration and dysregulation of c-Myc and mTOR (Fig. K). Moreover, we found a decrease in global transcription (Fig. L), consistent with the reduction of rRNAs that drives translation deficiency and halt of protein synthesis (Fig. M). All these data lead us to suggest the existence of a ribosomopathy, so we focused on the nucleolus, where the ribosome components are synthesized. Thus, Hnrnpk overexpressing cells showed a reduction in Fbl, increase in Ncl expression, condensates and Ncl diffusion in nucleoplasm. Summary/Conclusion Our work found that hnRNP K overexpression in vivo drives a BM failure phenotype, promoting the exhaustion of HSCs by ribosome dysregulation that triggers cell senescence. We therefore suggest that hnRNP K induces ribosome dysfunction consistent with some types of ribosomopathies.Keywords: Cell cycle, Senescence, Bone marrow failure